0013-7227/02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(12):5695–5701 Printed in U.S.A. Copyright © 2002 by The Endocrine Society doi: 10.1210/jc.2002-020970

11␤-Hydroxysteroid Dehydrogenase Types 1 and 2: An Important Pharmacokinetic Determinant for the Activity of Synthetic Mineralo- and

SVEN DIEDERICH, EKKEHARD EIGENDORFF, PATRICK BURKHARDT, MARCUS QUINKLER, CHRISTIANE BUMKE-VOGT, MARINA ROCHEL, DIETER SEIDELMANN, PETER ESPERLING, Downloaded from https://academic.oup.com/jcem/article/87/12/5695/2823639 by guest on 23 September 2021 WOLFGANG OELKERS, AND VOLKER BA¨ HR Department of Endocrinology, Diabetes, and Nutrition, Klinikum Benjamin Franklin, Freie Universita¨t Berlin (S.D., E.E., P.B., M.Q., C.B.-V., M.R., W.O., V.B.), and Research Laboratories of Schering AG (D.S., P. E.), 12200 Berlin, Germany

The 11␤-hydroxysteroid dehydrogenase (11␤-HSD) system Whereas the methyl groups also decrease reductase activity plays a pivotal role in (GC) and mineralocor- (steric effects), fluorination increases reductase activity (neg- ticoid (MC) action. Although 11␤-HSD activities are important ative inductive effect), leading to a shift to reductase activity. determinants for the efficacy of synthetic MCs and GCs, cor- This may explain the strong MC activity of 9␣-fluorocortisol responding pharmacokinetic data are scanty. Therefore, we and should be considered in GC therapy directed to 11␤-HSD2- characterized 11␤-HSD profiles for a wide range of expressing tissues (kidney, colon, and placentofetal unit). 11␤- often used in clinical practice. 11␤-HSD1 and 11␤-HSD2 were HSD2 oxidation of is more effective than that of selectively examined in 1) human liver and kidney cortex mi- , explaining the reduced MC activity of prednisolone crosomes, and 2) Chinese hamster ovarian cells stably trans- compared with cortisol. fected with 11␤-HSD1 or 11␤-HSD2 expression vectors. Both Reduction by 11␤-HSD1 is diminished by 16␣-methyl, 16␤- systems produced concordant evidence for the following methyl, 2␣-methyl, and 2-chlor substitution, whereas it is in- conclusions. creased by the ⌬1-dehydro configuration in , re- Oxidation of steroids by 11␤-HSD2 is diminished if they are sulting in higher hepatic first pass activation of prednisone fluorinated in position 6␣ or 9␣ (e.g. in ) or compared with . methylated at 2␣ or 6␣ (in ) or 16␣ or 16␤, To characterize a GC or a MC as substrate for the different by a methylene group at 16 (in ), methyloxazo- 11␤HSDs may be essential for an optimized therapy. line at 16, 17 (in ), or a 2-chlor configuration. (J Clin Endocrinol Metab 87: 5695–5701, 2002)

HE PRESENCE OF an 11␤-hydroxyl group (Fig. 1) is activity is an explanation for the strong MC activity of T essential for the antiinflammatory and immunosup- 9␣-fluorocortisol (13) and may be useful for targeted renal pressive effects of glucocorticoids (GCs) (1, 2) and for the immunosuppression (14). These examples show that consid- sodium-retaining effects of mineralocorticoids (MCs) (3). eration of the metabolism of synthetic GCs and MCs by Therefore, the interconversion of the 11␤-hydroxyl into the 11␤-HSDs may be very important for optimizing systemic corresponding 11␤-keto group and vice versa plays a pivotal GC and MC therapy. Therefore, we expand our previous role for the efficacy of these steroids (4). These reactions are examinations (15) on a large number of widely used synthetic catalyzed by the two enzymes 11␤-hydroxysteroid dehydro- steroids and characterized their substrate specificity for hu- genase type 1 (11␤-HSD1) and type 2 (11␤-HSD2). 11␤-HSD1 man 11␤-HSD1 and 11␤-HSD2. As human liver (11␤-HSD1) is expressed in a wide range of tissues (5). Whereas in vitro and kidney cortex (11␤-HSD2) selectively express these en- 11␤-HSD1 functions as a bidirectional enzyme (6), in vivo it zymes, incubations with microsome preparations from these works mainly as a reducing and activating enzyme (7). The organs are a valid and reproducible system for studying postulated function of 11␤-HSD1 seems to be autocrine or 11␤-HSDs (14). On the other hand, this system is somehow paracrine modulation of the GC status in nearly all GC target artificial, because the 11␤-HSD1 in isolated liver microsomes tissues (8). is not able to reduce cortisone to cortisol, which is the main 11␤-HSD2 is only found in MC target tissues (kidney, and most important reaction in vivo and is also observed in colon, and salivary glands) and the placenta, and exclusively tissue slices and whole cells. Therefore, we complemented oxidizes physiological GCs (9). The well defined physiolog- our experiments by using intact Chinese hamster ovarian ical function of 11␤-HSD2 is the protection of MC receptors (CHO) cells selectively transfected with human 11␤-HSD1 or from cortisol, thus providing MC selectivity to aldosterone (10). 11␤-HSD2 (16). In contrast to endogenous glucocorticoids, 9␣-fluorinated GCs show weak oxidase but strong reductase activity with Materials and Methods 11␤-HSD2 (11, 12). This 9␣-fluor induced shift to reductase Synthetic steroids Abbreviations: CHO, Chinese hamster ovarian; EMEM, Earle’s MEM; The following steroids were used as 11␤-HSD substrates. Cortisol (Fig. GC, glucocorticoid; 11␤-HSD, 11␤-hydroxysteroid dehydrogenase; MC, 1a), 2␣-methyl-cortisol, prednisolone (1,4-pregnadien-11␤,17,21-triol-3,20- mineralocorticoid; PCS, poly cloning site. dione; Fig. 1b), dexamethasone (9␣-fluoro-16␣-methyl-1,4-pregnadien-

5695 5696 J Clin Endocrinol Metab, December 2002, 87(12):5695–5701 Diederich et al. • 11␤-HSDs and Synthetic Steroids Downloaded from https://academic.oup.com/jcem/article/87/12/5695/2823639 by guest on 23 September 2021

FIG. 1. Steroid skeleton of cortisol (a) and positional modifications of some tested steroids. b, Prednisolone differs from cortisol by a ⌬1-dehydro configuration. c, Dexamethasone has additional 9␣-fluoro and 16␣- methyl groups compared with prednisolone. d, Deacetyl-deflazacort has an additional 16,17-methy- loxazoline group compared with prednisolone. e, Fluo- cortolone differs from dexamethasone in having the fluoro group at the 6␣ position and in bearing the 17-desoxy configuration of .

11␤,17,21-triol-3,20-dione; Fig. 1c), (9␣-fluoro-16␤-methyl- 1d was purchased from Hoechst Marion Roussel, Inc. (Bad Soden, Ger- 1,4-pregnadien-11␤,17,21-triol-3,20-dione), (16,17-butilidene- many). Deflazacort is deacetylated in a first pass metabolism, so that bis[oxy]-1,4-pregnadien-11␤,21-dihydroxy-3,20-dione), and 9␣-fluoro- deacetyl-deflazacort is the systemically available metabolite (18). If not cortisol (9␣-fluoro-11␤,17,21-trihydroxy-4-pregnen-3,20-dione) were ac- available, the corresponding 11-oxo-steroids were synthesized by oxi- quired from Sigma (St. Louis, MO). 6␣-Methyl-prednisolone (6␣-methyl- dation with chromium VI oxide (11). All steroids were purified by HPLC 1,4-pregnadien-11␤,17,21-triol-3,20-dione), 16␣-methyl-prednisolone before use. (16␣-methyl-1,4-pregnadien-11␤,17,21-triol-3,20-dione), 16␤-methyl- ␤ ␤ ␣ prednisolone (16 -methyl-1,4-pregnadien-11 ,17,21-triol-3,20-dione), 6 - Microsomes fluoro-cortisol (6␣-flouoro-11␤,17,21-trihydroxy-4-pregnen-3,20-dione), (6␣-flouoro-16␣-methyl-1,4-pregnadien-11␤,21-dihydroxy- Human liver and kidney cortex microsomes were prepared as pre- 3,20-dione; Fig. 1e), (6␣,9␣-diflouoro-16␣-methyl-1,4- viously described (14). Protein was quantified before every incubation pregnadien-11␤,21-dihydroxy-3,20-dione), and 2-chloro-fluocortolone using Bradford analysis. Substrate concentrations were chosen in the Ϫ (2-chloro-6␣-flouoro-16␣-methyl-1,4-pregnadien-11␤,21-dihydroxy- region of maximum velocity [kidney cortex (11␤-HSD2), 10 6 mol/liter, 3,20-dione) were all purchased from Schering AG (Berlin, Germany). liver (11␤-HSD1), 10–5 mol/liter]. The cosubstrate concentration (kid- (9␣-fluoro-1,4-pregnadien-11␤,17,21-triol-3,20-dione), 2␣- ney: NAD for oxidation, NADH for reduction; liver: NADP for oxida- Ϫ methyl-9␣-fluoro-cortisone (2␣-methyl-9␣-fluoro-17,21-dihydroxy-4- tion, NADPH for reduction) was 10 3 mol/liter. The pH for oxidation pregnen-3,11,20-trione), desoxymetasone (9␣-fluoro-16␣-methyl-1,4- was 8.5, and that for reduction was 6.0. For each steroid and each reaction pregnadien-11␤,21-diol-3,20-dione), and flumethasone (6␣,9␣-fluoro-16␣- tested, pilot studies for time and protein kinetics were performed so that methyl-1,4-pregnadien-11␤,17,21-triol-3,20-dione) were obtained from initial velocities could be measured in the linear range. Paesel and Lorei (Hanau, Germany). 2␣-Methyl-9␣-fluoro-cortisone was a special synthesis ordered by our group. As we mainly needed it for testing Transfected CHO cells reductase activity (17), we have ordered the synthesis of the 11-oxo form. Beclomethasone (9␣-chloro-16␤-methyl-1,4-pregnadien-11␤,17,21- The vector p11␤HSD1 was created using pSKHSD1, which was a gift triol-3,20-dione) was purchased from Glaxo Wellcome GmbH (Bad from Dr. A. K. Agarwal (19). The insert coding for the 11␤HSD1 was Oldesloe, Germany), prednylidene (16-methylene-1,4-pregnadien- excised from pSKHSD1 with HindIII and XbaI and ligated into the poly 11␤,17,21-triol-3,20-dione) was obtained from Merck & Co., Inc. (Darm- cloning site (PCS) of the mammalian expression vector pcDNA3.1 (In- stadt, Germany), (6␣-flouoro-11,16␣,17,21-tetrahydroxy-1,4- vitrogen, San Diego, CA) to produce p11␤HSD1. Before ligation the PCS pregnadien-3,20-dione-16,17-acetonid) was obtained from Boehringer was treated with HindIII and XbaI, and the small cleavage product Ingelheim GmbH (Ingelheim, Germany), and deacetyl-deflazacort was removed. For creation of p11␤HSD2, the expression plasmid (16,17-methyloxazoline-1,4-pregnadien-11␤,17,21-triol-3,20-dione; Fig. pcDNA1.1HSD2 coding for the 11␤-HSD2 (20) from Dr. A. K. Agarwal Diederich et al. • 11␤-HSDs and Synthetic Steroids J Clin Endocrinol Metab, December 2002, 87(12):5695–5701 5697

was cut with BamHI and NotI. The cleavage was ligated into the PCS of hydroxy and 11-oxoderivatives was performed using UV detection after pcDNA3.1 using the cleavage sites for BamHI and NotI. extraction by Sep-Pak C18 cartridges (Waters Corp., Milford, MA) and CHO-K1 cells (American Type Culture Collection, Manassas, VA) separation by reverse phase HPLC (22). In all incubations (microsomes were cultured in Ham’s F-12 medium (with stable glutamine derivative; and CHO cells), we were unable to detect any other metabolites than the Biochrome Berlin, Germany) supplemented with 10% fetal bovine se- 11-oxo- or 11-hydroxysteroids. Calculation of the percent conversion rum, penicillin (5000 U/ml), streptomycin (5000 ␮g/ml), and ampho- rates allowed determination of the initial velocities. tericin B (2.5 ␮g/ml). Cells were grown in six-well tissue culture plates to 60–70% confluence (37 C, 5% CO2). Transfection was performed using Statistics Lipofectamine (Life Technologies, Inc., Karlsruhe, Germany): 1 ␮g DNA ␤ ␤ ␮ (p11 HSD1 or p11 HSD2) was diluted in 100 l OptiMEM reduced Statistical calculations were performed using the SPSS program from ␮ serum medium (Life Technologies, Inc.), and 4 l Lipofectamine were SPSS, Inc. (Chicago, IL). An unpaired t test was used. diluted in 100 ␮l Earle’s MEM (EMEM) with 2 mm proline, without serum and antibiotics. Both solutions were combined and mixed gently. After 30 min, 800 ␮l EMEM with 2 mm proline without serum and Results

antibiotics were added to the lipid-DNA complexes, and this suspension 11␤-HSD2: steroid metabolism in human kidney cortex Downloaded from https://academic.oup.com/jcem/article/87/12/5695/2823639 by guest on 23 September 2021 was added to the washed cells. After 5-h incubation (37 C, 5% CO2), 1 microsomes (Table 1) and by transfected CHO cells (Figs. 2 ml EMEM containing 10% fetal bovine serum and 2 mm proline was added. After 72 h Ham’s F-12 medium with the additives described and 3) ␮ above and an additional 400 g/ml geniticin (Life Technologies, Inc.) Whereas oxidation by 11␤-HSD2 was the sole reaction for was added to select transfected cells. Selected clones were propagated in the same medium, but with reduced geniticin (200 ␮g/ml). unfluorinated steroids, fluorinated steroids were metabo- Incubations were performed in flasks with a 75-cm2 culture area lized bidirectionally, with a strong preference for reductase (Falcon 3084, BD Biosciences, Heidelberg, Germany). For each reaction activity (Table 1). tested, pilot studies were performed to determine optimal conditions. ␤ ϫ For 11 -HSD2 oxidation, the incubation volume was 13 ml with 2.7 ␤ 106 cells, the incubation time was 12 h, and the substrate concentration 11 -HSD2-oxidation Ϫ was 10 6 mol/liter. For each experiment, cortisol served as the control ␤ ␣ ϭ Oxidation by 11 -HSD2 was diminished by 6 -methyl, steroid (percent conversion, 23.7–46.5%; n 27; mean, 34.2%; sd, 5.7%). ␣ ␤ ␣ ␣ For 11␤-HSD2 reduction, the incubation volume was 13 ml with 27 ϫ 16 -methyl, 16 -methyl, and 6 -fluor or 9 -fluor substitu- 106 cells, the incubation time was 12 h, and the substrate concentration tion (Table 1, footnotes a and b). In addition, a 2␣-methyl Ϫ was 10 6 mol/liter. For 11␤-HSD1 reduction, the incubation volume was group (cortisol vs. 2␣-methyl-cortisol), a 16,17-methyloxazo- 13 ml with 2.7 ϫ 106 cells, the incubation time was 24 h, and the substrate Ϫ line configuration (prednisolone vs. deacetyl-deflazacort, concentration was 10 5 mol/liter. In each experiment cortisone served as the control steroid (percent conversion, 12.5–28.0%; n ϭ 19; mean, Fig. 1d), a 16-methylene substitution (prednisolone vs. pred- 18.9%; sd, 4.6%). nylidene), and a 2-chlor substitution (fluocortolone vs. 2-chlor-fluocortolone) also decreased 11␤-HSD2 oxidation Analysis of 11␤-HSD activity (Fig. 2). Both topically used GCs tested showed little oxida- ␤ Steroid extraction, separation, and quantitation were performed as tion by 11 -HSD2 (budesonide and flunisolide, Fig. 2). Mul- described previously (21). As the steroids tested were only available tiple substitutions in these steroids did not allow systematic as nonradioactive substrates, quantification of the corresponding 11- comparison with the other steroids tested.

TABLE 1. Metabolism of synthetic steroids by 11␤-HSD2 of human kidney cortex microsomes

Steroid Structure V0 oxidation V0 reduction Unfluorinated steroids Cortisol 29.3 Ϯ 2.2a Prednisolone ⌬1-Dehydro-cortisol 57.5 Ϯ 3.2b 6␣-Methyl-prednisolone 16.2 Ϯ 1.9 16␣-Methyl-prednisolone 27.2 Ϯ 2.3 16␤-Methyl-prednisolone 38.7 Ϯ 1.9 Fluorinated steroids 6␣-Fluorocortisol 19.0 Ϯ 1.0 88.2 Ϯ 15.0 9␣-Fluorocortisol 21.0 Ϯ 1.2 100.7 Ϯ 8.1 Isoflupredone 9␣-Fluoro-prednisolone 21.5 Ϯ 0.3 462.5 Ϯ 81.9c Dexamethasone 16␣-Methyl-9␣-fluoro-prednisolone 15.3 Ϯ 1.1 230.6 Ϯ 22.0d Betamethasone 16␤-Methyl-9␣-fluoro-prednisolone 24.3 Ϯ 1.6 149.3 Ϯ 23.0 Flumethasone 16␣-Methyl-6␣,9␣-fluoro-prednisolone 19.0 Ϯ 2.2 376.4 Ϯ 24.8 Desoxymetasone 17-Desoxy-dexamethasone 15.4 Ϯ 2.0 218.8 Ϯ 18.2 Diflucortolone 17-Desoxy-flumethasone 11.8 Ϯ 0.6 709.7 Ϯ 30.9e Fluocortolone Diflucortolone without 9␣-fluoro-group 52.5 Ϯ 4.2f,g 11.2 Ϯ 0.8h ϭ ⅐ Ϯ Initial velocities (V0 pmol/mg min) were measured, and six replicates were done for each reaction. Data are expressed as means SD. Substrate concentration: 10Ϫ6 mol/liter; cosubstrate concentration (NAD for oxidation, NADH for reduction): 10Ϫ3 mol/liter, pH for oxidation 8.5, for reduction 6.0. Reductase activity was not to detect with unfluorinated steroids. a Ͻ ␣ ␣ P 0.001 compared with V0 oxidation of 6 - and 9 -fluorocortisol. b Ͻ ␣ ␣ ␤ P 0.001 compared with V0 oxidation of cortisol and of 6 -methyl-, 16 -methyl-, and 16 -methyl-prednisolone. c Ͻ ␣ P 0.001 compared with V0 reduction of 9 -fluorocortisol and dexamethasone. d Ͻ P 0.001 compared with V0 reduction of betamethasone and flumethasone. e Ͻ P 0.001 compared with V0 reduction of desoxymetasone and fluocortolone. f Ͻ P 0.001 compared with V0 oxidation of all other fluorinated steroids and of cortisol. g Ͻ P 0.05 compared with V0 oxidation of prednisolone. h Ͻ P 0.001 compared with V0 reduction of all other fluorinated steroids. 5698 J Clin Endocrinol Metab, December 2002, 87(12):5695–5701 Diederich et al. • 11␤-HSDs and Synthetic Steroids Downloaded from https://academic.oup.com/jcem/article/87/12/5695/2823639 by guest on 23 September 2021

FIG.3.11␤-Reduction (Ⅺ)of9␣-fluorocortisone and 2-methyl-9␣- FIG.2.11␤-Oxidation (f) of unfluorinated and fluorinated steroids fluoro-cortisone by 11␤-HSD2-transfected CHO cells. Initial veloci- by 11␤-HSD2-transfected CHO cells. Initial velocities (Vo; mean Ϯ SD) ties (Vo; mean Ϯ SD) are presented. Six replicates were performed for are presented. Six replicates were performed for each reaction. The both reactions. The substrate concentration was 10Ϫ6 mol/liter; in- Ϫ substrate concentration was 10 6 mol/liter; incubation was for 12 h; cubation was for 12 h; 27 ϫ 106 cells in 13 ml medium were used. **, 2.7 ϫ 106 cells in 13 ml medium were used. n.c., No conversion. **, P Ͻ P Ͻ 0.01. 0.01; ***, P Ͻ 0.001. Reductase activity was not measured for these steroids. For steroid structures, see Fig. 1, Table 1, and the text. sition 6, as in fluocortolone (Fig. 1e), combined with the Prednylidene has an additional 16-methylene group compared with 17-desoxy configuration led to an impressing increase in prednisolone. Budesonide and flunisolide are topically used steroids oxidase activity (Table 1, footnote f) and a strong decrease in with complex structures (see the text). reductase activity (Table 1, footnote h). Therefore, fluocor- 11␤-HSD2 oxidation was increased by a ⌬1-dehydro con- tolone is unique in the group of fluorinated steroids, because figuration (prednisolone vs. cortisol; Table 1, footnote b, and it has its redox equilibrium on the inactive 11-oxo side, as do Fig. 2). In addition, fluocortolone (Fig. 1e; 17-desoxy config- the unfluorinated steroids (Table 1). ␣ In concordance with effects on 11␤-HSD2 oxidation, a uration and 6 -fluor substitution) showed surprisingly high ␤ oxidative activity in the group of fluorinated steroids (Table substitution with chlorine in position 2 totally abolished 11 - 1, footnote f, and Fig. 2). HSD2 reductase activity (experiments with 11-dehydro-2- chlor-fluocortolone in 11␤-HSD2-transfected CHO cells; data 11␤-HSD2 reduction not shown).

Reduction by 11␤-HSD2 was decreased by 2␣-methyl (Fig. 11␤-HSD1: steroid metabolism in human liver microsomes 3), 16␣-methyl, and 16␤-methyl substitution (dexamethasone (Table 2) and by transfected CHO cells (Fig. 4) and betamethasone vs. isoflupredone, Table 1). ␤ Whereas 6␣-or9␣-fluorination decreased 11␤-oxidation, it In agreement with the effects on 11 -HSD2, the redox ␤ equilibrium of 11␤-HSD1 is also shifted to the active 11- dramatically increased 11 -reduction, leading to a shift of the ␣ ␣ 11␤-HSD2 redox equilibrium to the active 11-hydroxy side hydroxy side by 6 -or9 -fluorination (Table 2 and Fig. 4, ␣ ␣ cortisol vs. 9␣-fluoro-cortisol). In human liver microsomes, (Table 1). 6 - and 9 -fluorination had additive effects in ␤ augmenting reductase activity (flumethasone vs. dexameth- 11 -reduction could only be demonstrated with fluorinated asone, diflucortolone vs. desoxymetasone; Table 1). In con- steroids (Table 2), whereas transfected CHO cells also cordance with the effects on 11␤-oxidation, the ⌬1-dehydro showed reductase activity with unfluorinated steroids such configuration also increased 11␤-reduction (isoflupredone as cortisone or prednisone (Fig. 4). vs. 9␣-fluoro-cortisol, Table 1). 11␤-HSD1 oxidation The effects of the 17-desoxy configuration seem to be in- fluenced by other modifications in the structure of the ste- Oxidation by 11␤-HSD1 was decreased by a 16␣-methyl roid. In double fluorinated steroids such as flumethasone, it group (Table 2, footnote a) and was abolished by 6␣-fluor or increased reductase activity and decreased oxidase activity, 9␣-fluor substitution (Table 2, all fluorinated steroids). In leading to a shift to the active 11-hydroxy side (flumethasone contrast to 11␤-HSD2 oxidation (Table 1), 6␣-methyl and vs. diflucortolone, Table 1). In monofluorinated steroids in 16␤-methyl groups showed no significant effect on 11␤- position 9, it had no significant effects (dexamethasone vs. HSD1 oxidation (Table 2). Oxidation by 11␤-HSD1 was in- desoxymetasone, Table 1), whereas monofluorination in po- creased by the ⌬1-dehydro configuration (Table 2, footnote a). Diederich et al. • 11␤-HSDs and Synthetic Steroids J Clin Endocrinol Metab, December 2002, 87(12):5695–5701 5699

TABLE 2. Metabolism of synthetic steroids by 11␤-HSD1 of human liver microsomes

Steroid Structure V0 oxidation V0 reduction Unfluorinated steroids Cortisol 101.4 Ϯ 10.0 Prednisolone ⌬1-Dehydro-cortisol 136.7 Ϯ 11.6a 6␣-Methyl-prednisolone 138.6 Ϯ 17.8 16␣-Methyl-prednisolone 72.2 Ϯ 7.1 16␤-Methyl-prednisolone 143.1 Ϯ 11.7 Fluorinated steroids 6␣-Fluorocortisol 218.9 Ϯ 20.8 9␣-Fluorocortisol 260.6 Ϯ 30.9b Isoflupredone 9␣-Fluoro-prednisolone 1175.0 Ϯ 105.3c Dexamethasone 16␣-Methyl-9␣-fluoro-prednisolone 540.0 Ϯ 55.3d Betamethasone 16␤-Methyl-9␣-fluoro-prednisolone 377.8 Ϯ 34.2 Downloaded from https://academic.oup.com/jcem/article/87/12/5695/2823639 by guest on 23 September 2021 Flumethasone 16␣-Methyl-6␣,9␣-fluoro-prednisolone 650.0 Ϯ 84.3e Diflucortolone 17-Desoxy-flumethasone 1338.9 Ϯ 139.3f ϭ ⅐ Ϯ Initial velocities (V0 pmol/mg min) were measured, and six replicates were done for each reaction. Data are expressed as means SD. Substrate concentration: 10Ϫ5 mol/liter; cosubstrate concentration (NADP for oxidation, NADPH for reduction): 10Ϫ3 mol/liter, pH for oxidation 8.5, for reduction 6.0. Whereas reductase activity was not to detect with unfluorinated steroids, fluorinated steroids show no oxidase activity. a Ͻ ␣ P 0.001 compared with V0 oxidation of cortisol and of 16 -methyl-prednisolone. b Ͻ ␣ P 0.05 compared with V0 reduction of 6 -fluorocortisol. c Ͻ ␣ P 0.001 compared with V0 reduction of 9 -fluorocortisol and dexamethasone. d Ͻ P 0.05 compared with V0 reduction of betamethasone. e Ͻ P 0.001 compared with V0 reduction of dexamethasone. f Ͻ P 0.001 compared with V0 reduction of flumethasone. 2␣-methyl-9␣-fluoro-cortisol and fluocortolone vs. 2-chlor- fluocortolone) led to a significant decrease in 11␤-HSD1 re- duction, 16-methylene and 6␣-methyl substituents had no effect on 11␤-HSD1 reduction (Fig. 4, prednisolone vs. pred- nylidene and 6␣-methyl-prednisolone). 11␤-HSD1 reduction was increased by 6␣-fluor or 9␣-fluor substitution (Table 2; all fluorinated steroids) and by the ⌬1-dehydro configuration (Table 2, isoflupredone vs. 9␣- fluoro-cortisol; Fig. 4, cortisol vs. prednisolone). 9␣-Chlor substitution instead of 9␣-fluor substitution significantly augmented the reductase activity of 11␤-HSD1 (beclometha- sone vs. betamethasone, Fig. 4). In addition, double fluori- nation in positions 6␣ and 9␣ (flumethasone vs. dexameth- asone, Table 2) as well as the 17-desoxy configuration (diflucortolone vs. flumethasone, Table 2) led to increased reductase activity of 11␤-HSD1.

Discussion Apart from pharmacodynamic parameters like receptor binding and transactivation, the activity of a hormone is determined by pharmacokinetic characteristics. The most

FIG.4.11␤-Reduction (Ⅺ) of unfluorinated and fluorinated steroids important pharmacokinetic systems for GCs and MCs are the by 11␤-HSD1-transfected CHO cells. Initial velocities (Vo; mean Ϯ SD) 11␤-HSDs, because they regulate the target cell adjustment are presented. Six replicates were performed for each reaction. The between the active hydroxy and the inactive oxo form of a substrate concentration was 10Ϫ5 mol/liter; incubation was for 24 h; 6 steroid (4). In mammalian tissues at least two isoenzymes of 2.7 ϫ 10 cells in 13 ml medium were used. n.c., No conversion. **, P Ͻ ␤ 0.01; ***, P Ͻ 0.001. The 11-hydroxysteroids are listed; the 11-oxo- 11 -HSD are responsible for this elegant autocrine fine- steroids were the substrates for incubation. Oxidase activity was not tuning. Whereas the widely distributed 11␤-HSD1 acts pre- measured for these steroids. For steroid structures, see Fig. 1, Table dominantly as a reductase, facilitating GC hormone action, 1, and the text. Prednylidene has an additional 16-methylene group the selectively expressed 11␤-HSD2 acts as an oxidizing en- compared with prednisolone. Beclomethasone differs from beta- in having a 9␣-chloro group instead of the 9␣-fluoro group. zyme, thus diminishing the GC and MC activities of a steroid in the corresponding organs. Beside this autocrine function, 11␤-HSD1 has an important pharmacological function, be- 11␤-HSD1 reduction cause hepatic 11␤HSD1 activates orally given pro-drugs such Whereas 16␣-methyl and 16␤-methyl groups (Table 2, iso- as cortisone or prednisone to their active 11-hydroxy deriv- flupredone vs. dexamethasone and betamethasone) and 2␣- atives (23, 24). methyl and 2-chlor substitutions (Fig. 4, 9␣-fluoro-cortisol vs. In this context the knowledge about 11␤-HSD substrate 5700 J Clin Endocrinol Metab, December 2002, 87(12):5695–5701 Diederich et al. • 11␤-HSDs and Synthetic Steroids specificity of a GC or a MC seems to be indispensable for an seems to result from combination with the 17-deoxy config- optimized steroid therapy. Moreover, as fluorinated steroids uration. In the group of unfluorinated steroids, prednisolone are mainly reduced by 11-␤HSD2, we have developed the shows the highest activity for 11␤-HSD2 oxidation. Concern- idea of targeted renal immunosuppression with a fluorinated ing GC therapy, 11-hydroxysteroids with high inactivation 11-dehydrosteroid, which should have high affinity to 11␤- by 11␤-HSD2 (e.g. fluocortolone and prednisolone) should be HSD2, but low affinity to 11␤-HSD1 (14). As both isoenzymes used when the 11␤-HSD2-expressing tissues have to be pro- share only 14% homology, divergent effects of modifications tected from GC action, e.g. in pregnancy when the mother in the steroid structure can be expected. and not the fetus needs GC treatment. Human kidney cortex and liver microsomes as well as Apart from the discussed recommendations for GC ther- selectively transfected CHO cells are both well established apy, our results allow some conclusions concerning the ac- systems for studying 11␤-HSD activity. Although the initial cess of 11-hydroxysteroids to the MC receptor, which is the velocities of the different systems cannot be compared be- physiolocically most important function of 11␤-HSD2. The Downloaded from https://academic.oup.com/jcem/article/87/12/5695/2823639 by guest on 23 September 2021 cause the concentrations of the 11␤-HSDs are not known, increased 11␤-HSD2 oxidation of prednisolone compared comparison between different steroids in the same system is with cortisol may lead to enhanced renal inactivation (30), a useful method to determine the effects of specific substi- thus giving a good pharmacokinetic explanation for the re- tutions on the metabolism by 11␤-HSDs. Moreover, the high duced MC activity of prednisolone compared with cortisol. agreement of the results in both systems underlines the cor- On the other hand, the diminished 11␤-HSD2 oxidation of rectness of our data. 9␣-fluoro-cortisol is one explanation for its strong MC ac- Oxidase activity of 11␤-HSD2 seems to be very important tivity compared with cortisol (13). for GC therapy targeted to organs expressing mainly or ex- The most important pharmacological function of 11␤- clusively 11␤-HSD2 (kidney and colon). High 11␤-HSD2 ox- HSD1 is hepatic first pass activation of orally given inactive idation of an 11-hydroxy-GC should result in strong inacti- 11-dehydrosteroids (23, 24). The increased 11␤-HSD1 reduc- vation in the corresponding organs, whereas 11-hydroxy tion of prednisone compared with cortisone may explain GCs with low 11␤-HSD2 oxidation should have significant why orally given prednisone shows a more effective hepatic pharmacokinetic advantages because of their reduced inac- activation than cortisone, resulting in higher systemic avail- tivating capacity in the target tissues. As 11␤-HSD2 in the ability of the active 11-hydroxy form prednisolone (23). placenta protects the fetus from maternal GCs (25), the best Concerning our idea of renal GC targeting (14), we are option for intrauterine treatment of the fetus should be ste- interested in substituents that decrease 11␤-HSD1 reduction roids with very low oxidation by 11␤-HSD2. If the mother but do not affect 11␤-HSD-2 reduction. A fluorinated 11- and not the fetus needs GC therapy, steroids with high 11␤- dehydrosteroid with such a modification should pass 11␤- HSD2 oxidation should be the preference. HSD1 in the liver nearly unchanged, resulting in low plasma We have defined many substituents (2␣-methyl, 2-chlor, concentrations of the corresponding 11-hydoxysteroid and 6␣-methyl, 16␣-methyl, 16␤-methyl, 16-methylene, and thus minimizing systemic side-effects of the steroid. As renal 16,17-methyloxazoline substituents) that lead to steric inhi- 11␤-HSD2 activity determines local 11-hydroxy/11-dehydro bition of 11␤-HSD2 oxidation and may be favorable for GC ratios in the kidney (30), the fluorinated 11-dehydrosteroid therapy directed to 11␤-HSD2-expressing organs. The most should be specifically activated in the kidney, leading to a important effect of substitution is that of 6␣-or9␣-fluorina- targeted enrichment of the active 11-hydroxysteroid in this tion, which diminishes 11␤-HSD2 oxidation and dramati- organ. Similar to immunosuppression in inflammatory lung cally increases 11␤-HSD2 reduction, thus leading to a strong and skin diseases, local intrarenal manipulation of the im- shift to the active 11-hydroxy form of a steroid. Therefore, mune response seems to be the most important factor de- fluorinated 11-hydroxysteroids show the lowest inactivating termining renal allograft survival (31, 32). Therefore, our capacity by 11␤-HSD2 and should be the best therapeutic approach may be a promising way for renal immunosup- option for intrauterine treatment of the fetus or for renal pression with reduced side-effects. immunosuppression. As double fluorination in positions 6␣ Reduction by 11␤-HSD1 was diminished for 16␣-methyl-, and 9␣ augments this shift to the active 11-hydroxy side, 16␤-methyl-, and especially 2␣-methyl- and 2-chlor-substi- flumethasone or diflucortolone seems to be the first choice tuted steroids. A 2-chlor substitution totally inhibited 11␤- for these approaches. HSD1 reduction, but showed the same effect on oxidation The fluor-induced shift to reductase activity can be en- and reduction by 11␤-HSD2. As 2␣-, 16␣-, and 16␤-methyl- hanced by substituting chlorine for fluor in the 9␣ position ations also result in decreased reduction by 11␤-HSD2, these (beclomethasone vs. betamethasone). This underlines the modifications are not optimal for the idea of renal GC tar- thesis that the negative inductive effect of halogen atoms geting. Therefore, further investigations, preferentially with leads to the shift in the redox equilibrium (26). computer-designed substances, have to be performed. An Both topically used GCs tested (budesonide and flunisolide) alternative approach for GC targeting to the kidney may be show little inactivation by 11␤-HSD2. As the target organs of the combination of an available fluorinated 11-dehydro- topical GC therapy (lung, skin, and rectum) also express 11␤- steroid (e.g. 11-dehydro-dexamethasone or 11-dehydro- HSD2 (9, 27–29), a pharmacokinetic profile with little 11␤-HSD2 diflucortolone) with a selective inhibitor of 11␤-HSD1 (33). oxidation seems to be favorable for these substances. In summary, we have characterized the 11␤-HSD profile In the group of fluorinated steroids, fluocortolone is an of a wide range of clinically often used steroids. Both in vitro interesting exception, because this steroid, despite a 6␣-fluor systems used were shown to be suitable for this approach: substitution, shows effective oxidation by 11␤-HSD2, which 1) human kidney and liver microsomes (to measure 11␤-HSD1 Diederich et al. • 11␤-HSDs and Synthetic Steroids J Clin Endocrinol Metab, December 2002, 87(12):5695–5701 5701 reductase activity in microsomes, fluorinated steroids have to 9. Whorwood CB, Mason JI, Ricketts ML, Howie AJ, Stewart PM 1995 Detection ␤ ␤ of human 11␤-hydroxysteroid dehydrogenase isoforms using reverse-tran- be used) and 2) stably 11 -HSD1- and 11 -HSD2-transfected scriptase-polymerase chain reaction and localization of the type 2 isoform to CHO cells (which have the advantage of permanent availability renal collecting ducts. Mol Cell Endocrinol 110:R7–R12 and relatively high similarity to the in vivo conditions). 10. 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Assandri A, Buniva G, Martinelli E, Perazzi A, Zerilli L 1984 Pharmacoki- As prednisolone, the widely used GC for renal immuno- netics and metabolism of deflazacort in the rat, dog, monkey and man. Adv ␤ Exp Med Biol 171:9–23 suppression, shows very strong oxidation by 11 HSD2, this 19. Tannin GM, Agarwal AK, Monder C, New MI, White PC 1991 The human steroid does not seem to be ideal for these patients. Thus, gene for 11␤-hydroxysteroid dehydrogenase. Structure, tissue distribution, exact clinical studies comparing prednisolone with, for ex- and chromosomal localization. J Biol Chem 266:16653–16658 ␣ 20. Agarwal AK, Rogerson FM, Mune T, White PC 1995 Gene structure and ample, 6 -methyl-prednisolone, deflazacort, and dexameth- chromosomal localization of the human HSD11K gene encoding the kidney asone in kidney transplantation should be very informative. (type 2) isozyme of 11␤-hydroxysteroid dehydrogenase. Genomics 29:195–199 21. Quinkler M, Johanssen S, Grossmann C, Ba¨hr V, Muller M, Oelkers W, Diederich S 1999 metabolism in the human kidney and inhibi- Acknowledgments tion of 11␤-hydroxysteroid dehydrogenase type 2 by progesterone and its ␤ metabolites. J Clin Endocrinol Metab 84:4165–4171 We thank Dr. A. K. Agarwal for the 11 -HSD plasmids, and Drs. H. 22. Diederich S, Quinkler M, Miller K, Heilmann P, Scho¨nesho¨fer M, Oelkers Laurent and H. J. Zentel (Schering AG, Berlin, Germany) for assistance W 1996 Human kidney 11␤-hydroxysteroid dehydrogenase: regulation by in steroid preparation and separation. We also thank Mrs. G. Kainzbauer adrenocorticotropin? Eur J Endocrinol 134:301–307 for assistance with transfection studies. 23. Jenkins JS, Sampson PA 1967 Conversion of cortisone to cortisol and pred- nisone to prednisolone. Br Med J 2:205–207 Received June 25, 2002. Accepted September 6, 2002. 24. Stewart PM, Boulton A, Kumar S, Clark PMS, Shackleton CHL 1999 Cortisol metabolism in human obesity: impaired cortisone to cortisol conversion in Address all correspondence and requests for reprints to: Dr. Sven subjects with central adiposity. J Clin Endocrinol Metab 84:1022–1027 Diederich, Department of Endocrinology, Diabetes, and Nutrition, 25. Krozowski Z, Maguire JA, Stein-Oakley AN, Dowling J, Smith RE, Andrews Klinikum Benjamin Franklin, Freie Universita¨t Berlin, Hindenburg- RK 1995 Immunohistochemical localization of the 11␤-hydroxysteroid dehy- damm 30, 12200 Berlin, Germany. E-mail: [email protected]. drogenase type II enzyme in human kidney and placenta. J Clin Endocrinol This work was supported by a grant from Deutsche Forschungsge- Metab 80:2203–2209 sellschaft (DI 741/1-3). 26. Bush IE, Hunter SA, Meigs RA 1968 Metabolism of 11-oxygenated steroids. Metabolism in vitro by preparations of liver. Biochem J 107:239–258 27. 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